R848 (Resiquimod)

TLR7/TLR8 Agonist - Imidazoquinoline compound

TLR7/TLR8 activation with R848

R848 (Resiquimod), an imidazoquinoline, is a dual TLR7 and TLR8 synthetic agonist with potent antiviral activity [1-3]. It induces differential TLR7 and/or TLR8 responses in human and murine immune cells. TLR7 and TLR8 are endosomal pattern recognition receptors that play an important role in the antiviral immune response.

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Mode of action

R848 (Resiquimod), a low molecular weight synthetic molecule, acts as a selective activating ligand for both TLR7 and TLR8 in humans but only TLR7 in mice. It activates immune cells via the TLR7/TLR8 MyD88-dependent signaling pathway with the subsequent activation of the transcription factors NF-κB and interferon regulatory factor (IRF) [2, 3]. This ultimately leads to the production of pro-inflammatory cytokines and type I interferons. 

Unlike other commercially available R848 preparations, InvivoGen's R848 is water soluble, validated for TLR7/TLR8 potency, and tested to ensure the absence of TLR2 or TLR4 contamination. 

InvivoGen also offers R848 VacciGrade™, a high-quality pre-clinical grade suited for in vivo studies. 

Key features of R848

• Agonist of hTLR7 and hTLR8
• Agonist of mTLR7
• Each lot is highly pure (≥95%) and functionally tested
• High-quality, pre-clinical R848 VacciGrade™ is also available for in vivo studies

Read our review about TLR7 and TLR8.

References:

1. Vanwalscappel B. et al., 2018. Toll-like receptor agonist R848 blocks Zika virus replication by inducing the antiviral protein viperin. Virology 522:199-208.
2. Hemmi H. et al., 2002. Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat Immunol. 3(2):196-200.
3. Jurk M. et al. 2002. Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R848. Nat Immunol. 3(6):499.

Figures

NF-κB response of HEK-Blue™-derived cells to R848. HEK-Blue™ cells expressing hTLR7, mTLR7, hTLR8, or mTLR8 cultured in HEK-Blue™ Detection reagent and stimulated with increasing concentrations of R848. After 24h incubation, the NF-κB-induced SEAP activity was assessed by measuring the SEAP level in the supernatant. Data are shown as optical density (OD) at 650 nm (mean ± SEM). Of note, HEK-Blue™ Null* comprises data from parental cell lines HEK-Blue Null1, HEK-Blue Null1-v, HEK-Blue Null2-k.

NF-κB and IRF responses of THP1-Dual™-derived cells to R848. THP1-Dual™, THP1-Dual™ hTLR7 cells, and THP1-Dual™ hTLR8 cells were incubated for 24 hours with increasing concentrations of R848. After 24h incubation, the (A) NF-κB-induced SEAP activity was assessed using QUANTI-Blue™. Data are shown as optical density (OD) at 650 nm (mean ± SEM). (B) The IRF response was assessed by measuring the activity of Lucia luciferase in the supernatant using QUANTI-Luc™. Data are shown in fold response over non-induced cells (mean ± SEM).

Specifications

Specificity: Human TLR7, human TLR8, and murine TLR7

Working Concentration: 10 ng - 10 μg/ml

CAS number: 144875-48-9 (free base)

Solubility: 1 mg/ml in water

Formula: C17H22N4O2 • HCl

Molecular weight: 350.8 g/mol

Quality control:

• Purity: ≥95% (UHPLC)
• The activation of human TLR7, human TLR8, and murine TLR7 by R848 has been confirmed using cellular assays.
• The absence of bacterial contamination (e.g. lipoproteins and endotoxins) has been confirmed using HEK-Blue™ hTLR2 and HEK-Blue™ hTLR4 cells.

Contents

R848 (Resiquimod) is provided lyophilized and is available in two quantities:

• tlrl-r848-1: 2 x 500 µg and 1.5 ml of sterile endotoxin-free water
• tlrl-r848-10: 2 x 5 mg and 10 ml of sterile endotoxin-free water

R848 is shipped at room temperature.

Upon receipt, it should be stored at 4°C or -20°C.

Details

TLR7 and TLR8:

TLR7 and TLR8 are endosomal pattern recognition receptors that share structural homology [1]. Both receptors are activated by single-stranded RNA (ssRNA) molecules, however, they exhibit different ligand-binding specificities and cellular expression patterns suggesting that they have nonredundant specialized roles.

TLR7 is essentially expressed by plasmacytoid dendritic cells (pDCs) but is also found in B cells and other myeloid cells [2] while TLR8 is highly expressed by myeloid cells and is absent from pDCs and B cells [2]. 

The endosomal distribution of TLR7 and TLR8 allows them to scan for the presence of microbial RNA in the phagocytic cargo. Their activation leads to NF-κB-, AP1-, and interferon regulatory factor (IRF)-mediated production of type I interferons (IFN-α/β) and pro-inflammatory cytokines [2].

Structural analyses have revealed that both TLR7 and TLR8 possess two binding sites (designated as Site 1 and Site 2) which do not share the same specificities.

Site 1 is highly conserved between TLR7 and TLR8 and binds nucleosides (guanosine (G) for TLR7 and uridine (U) for TLR8) or base analogs. The ligand preference for TLR7 and TLR8 is thus explained by the presence of specific residues in Site 1. Site 1 occupancy allows receptor dimerization and signaling.

Site 2 is less conserved and binds ssRNA with U(U) and U(G) motifs, respectively [3, 4]. Of note, ssRNA-binding to Site 2 is not sufficient for the formation of a signaling-competent TLR dimer but it strongly enhances the binding affinity of Site 1 [3, 4]. Thus, TLR7 and TLR8 appear to sense distinct RNA-degradation products rather than full-length ssRNAs [4].

1. Chuang T.H. & Ulevitch R.J., 2000. Cloning and characterization of a sub-family of human toll-like receptors: hTLR7, hTLR8, and hTLR9. Eur Cytokine Netw, 11:372-8.
2. Georg P. & Sander L.E., 2019. Innate sensors that regulate vaccine responses. Curr. Op. Immunol. 59:31.
3. Zhang Z. et al., 2018. Structural analyses of Toll-like receptor 7 reveal detailed RNA sequence specificity and recognition mechanism of agonistic ligands. Cell Rep. 25:3371.
4. Tanji H. et al., 2015. Toll-like receptor 8 senses degradation products of single-stranded RNA. Nat. Struct. Mol. Biol. 22:109.

Structure of R848 (Resiquimod):